The present invention relates to novel carboxyalkyl 1-position derivatives of lysergic acid diethylamide (LSD) and methods for preparation of these derivatives. The derivatives include immunogens used to stimulate antibody production and polypeptide conjugates useful in immunoassays for detecting LSD. Also provided are hapten intermediates used in the synthesis of the immunogens and polypeptide conjugates and a non-isotopic immunoassay for the determination of LSD.
Although there is widespread public perception that use of LSD is no longer a societal problem, there is considerable evidence that this illicit drug continues to be used, and in some segments of the population, its use is increasing. See Bonner, Drug Detection Report. 1:5 (1992). LSD was one of the 20 controlled substances most commonly encountered in emergency rooms across the nation in 1985, reflecting continuing abuse and trafficking of this illicit drug. In the United States, seizures of LSD by the Drug Enforcement Agency doubled in 1990 over the previous year, and in England, seizures of LSD have steadily increased since mid-1988. See Microgram 23:228 (1990). Further cause for concern are reports that LSD is particularly popular among adolescents, and in some areas, it exceeds cocaine in popularity. See Seligmann, Newsweek, February 3rd, p. 66, (1992). Factors that have contributed to the continued use of LSD are its wide availability, low cost, and the difficulty of detecting LSD use by analysis of body fluids.
Despite the long history of abuse associated with LSD, little is known concerning the disposition of LSD in humans. The lack of pharmacokinetic data on LSD is partly due to the technical difficulty of detecting and measuring the drug in physiological specimens. LSD is not considered highly toxic, although at least two cases where death was apparently a result of LSD toxicity have been reported. However, the major reason many consider LSD to be highly dangerous is that it can have serious psychological and psychotic effects which sometimes cause users to commit irrational acts resulting in injury or death. LSD is an extremely potent psychedelic drug that acts primarily on the central nervous system; only the d-isomer of the drug is pharmacologically active. Oral doses as low as 25 .mu.g can cause central nervous system disturbances such as hallucinations, distortions in sensory perception, mood changes and dream-like thought processes, as well as psychotic reactions in apparently predisposed individuals. Therefore, concentrations of LSD and LSD metabolites in blood and urine are likely to be very low. The detection of LSD in body fluids of users is especially difficult because the quantities typically ingested are very small and because the drug is rapidly and extensively converted to metabolic products. Furthermore, the drug's low volatility, its thermal instability, and its tendency to undergo adsorptive losses during gas chromatographic analysis all contribute to the difficulty of developing a method for confirmation of LSD in body fluids.
LSD is a natural product of the rye fungus Claviceps and was first prepared synthetically in 1938. Its psychological effects were discovered following accidental ingestion. Chemically, LSD is an ergot alkaloid and, like other compounds of this class, contains lysergic acid as the basis of its structure. Structurally similar to serotonin (5-hydroxytryptamine), LSD is thought to exert its psychotomimetic effects through antagonism of serotonin activity in the brain stem. Little is known about the tissue distribution, metabolism and excretion of LSD in humans. LSD is absorbed fairly rapidly by the gastrointestinal tract, and its plasma half-life has been calculated to be about 3 hours in man. Animal studies indicate that LSD is inactivated via hepatic oxidation. It is extensively metabolized with only negligible amounts of unchanged drug appearing in the urine and feces, with most of the metabolites being excreted in the urine. Possible metabolic transformations may be hydrolysis to lysergic acid, N-demethylation to nor-LSD and oxidation to 2-oxo-LSD. Studies with urine samples from human volunteers receiving LSD demonstrate that the drug or its closely related metabolites can be detected in the urine by radioimmunoassay (RIA) for several days following administration.
Although continued illicit use of LSD has stimulated efforts to develop effective analytical methods for the detection of the drug and its metabolites in body fluids from suspected LSD users, the methods currently available are complicated, time-consuming, expensive to perform and plagued by other problems. These methods include high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS) and radioimmunoassay. One problem faced by laboratories involved in the determination of LSD is the strong tendency for LSD and derivatized LSD to undergo adsorptive losses when subjected to gas chromatography. This behavior often prevents detection of the drug at the sub-nanogram/milliliter concentrations normally encountered in body fluids from LSD users.
Commercial RIAs for LSD are available from several sources, including ABUSCREEN LSD assay (.RTM. Roche Diagnostics Systems, Nutley, N.J.) and COAT-A-COUNT LSD assay (.RTM. Diagnostic Products Corp., Los Angeles, Calif.), and these products serve as a useful and relatively inexpensive method of screening for the presence of the drug. However, RIAs are not totally specific for LSD, so that an RIA-positive specimen still has to be confirmed by a second and more specific assay if the results of the analysis could have punitive consequences. The manufacturers' recommended cut-off concentration for considering a sample positive for LSD is 0.5 ng/ml, although lower cut-offs have been used in investigations where legal consequences were not a concern. The actual concentration of LSD in RIA-positive urine specimens is generally lower than that indicated by the RIA, and often considerably lower. Presumably the higher concentrations indicated by RIA are due to the cross-reactivity of LSD metabolites to the RIA antisera, but this conclusion cannot be substantiated until the major LSD metabolites in urine have been identified and their cross-reactivities determined.
In testing for other drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as .beta.-galactosidase and peroxidase, fluorescent molecules such as fluorescent compounds, and adioactive compounds such as .sup.125 I are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
One type of competitive binding immunoassay is based upon the reassociation of enzyymatically inactive polypeptide fragments to form active enzyme as a step of generating a detectable signal utilized to determine the amount of analyte present in a sample. This type of assay, known as cloned enzyme donor immunoassay (CEDIA), is described in U.S. Pat. No. 4,708,929. In particular, a .beta.-galactosidase enzyme donor polypeptide combines with a .beta.-galactosidase enzyme acceptor polypeptide to form active .beta.-galactosidase enzyme. Conjugating a hapten, or a small analyte or an analyte analog, to the enzyme donor polypeptide at certain sites does not affect the ability to form active .beta.-galactosidase by a complementation reaction and hence does not affect the rate of .beta.-galactosidase activity when in the presence of a substrate for .beta.-galactosidase. However, when the enzyme donor-hapten conjugate is bound by anti-analyte antibody, the complementation rate is impeded, and thereby the enzyme-catalyzed reaction rate during the initial phase of the reaction is reduced. This reduction in enzyme catalyzed reaction rate can be monitored and has been used successfully to determine a plurality of analytes using the principle of competitive inhibition whereby enzyme donor-analyte conjugate present in a reaction mixture and analyte present in a sample compete for anti-analyte antibody prior to the addition of enzyme acceptor. The complementation rate of .beta.-galactosidase formation, and hence enzyme-catalyzed reaction rate, is increased as the amount of analyte present in the sample is increased.
For the development of non-isotopic immunoassays which detect LSD and LSD metabolites, defined hapten derivatives are needed for preparation of immunogens and labeled conjugates. In particular, hapten derivatives at the 1-position of the lysergamide moiety are desirable because generally accepted strategy for preparing immunogens involves attachment of a hapten to the carrier substance at a position that is distant from the site in the molecule where immuno-specificity is desired, i.e., a site on the other side of the molecule from the ethyl side chains at the 8-position.
The preparation of antibodies to LSD for use in immunoassays to determine the drug has been accomplished in the prior art by several different approaches. One approach has been to couple the carboxyl group of lysergic acid directly to an immunogenic carrier protein, i.e. poly(L-lysine) or human serum albumin using carbodiimides. See Van Vunakis, Proc. Nat. Acad. Sci., 68:1483-87 (1971); Loeffler, J. Pharm. Sci. 62:1817-20 (1973); and Voss, Psychopharmacologia 26:140-45 (1972). This approach was used in developing early RIA methods for LSD determination, but the antibodies that were produced were characterized by poor specificity for LSD and high cross-reactivities with other ergot alkaloids.
A second approach has been to couple LSD to an immunogenic carrier protein via one of the ethyl side chains at the 8-position. See Ratcliffe, Clin. Chem. 23:169-74 (1977). In another approach, bis-diazo benzidine was used to couple the carrier proteins via an aromatic substitution. See Luderer, Bull. New Jersey Acad. Sci. 19:8-10 (1974).
Finally, LSD has been coupled to an immunogenic carrier protein via a linker arm using a reaction between LSD, formaldehyde and bovine serum albumin. See Castro, Res. Commun. Chem. Pathol. Pharmacol. 6:879-86 (1973); Taunton-Rigby, Science 181:165-6 (1973); and Ratcliffe, Clin. Chem. 23:169-74 (1977). These authors reported the reaction to be a Mannich aminoalkylation reaction, the product of which they postulated to be substituted at the N-1, or indole nitrogen, position. More recent investigation, however, based upon known condensation reactions of indole groups with an aldehyde, suggests that the 2-position was more probably the actual site of reaction. Several references describe the Mannich reaction with indole, and these references teach that no bond is formed at the N-1 position when there is an opportunity for reaction at another position on the indole molecule. When the 3-position and 2-position are both available, the site of reaction is at the 3-position, with the indole nitrogen remaining unreactive. This is described in Orchin, The Vocabulary of Organic Chemistry, John Wiley & Sons, N.Y., p. 385; Funiss, Vogel's Textbook of Practical Organic Chemistry, 4th Ed., Longman Scientific & Technical and John Wiley & Sons, N.Y., p. 813 (1978); and Mundy, Name Reactions and Reagents in Organic Synthesis, John Wiley & Sons, N.Y., p. 137 (1988). When the 3-position of the indole ring is blocked, however, as is the case in the LSD molecule, the reactive site involves the 2-position, with no bond being formed at the indole nitrogen, N-1. This is described in Orchin, The Vocabulary of Organic Chemistry, John Wiley & Sons, NY, p. 501, FIG. 13.790. Orchin describes the indole alkaloid tryptamine undergoing a Mannich condensation reaction with an aldehyde (secologanin), with the resulting substitution occurring at the 2-position on the indole group.
The LSD derivatives of the present invention are carboxyalkyl derivatives and, in contrast to prior art derivatives, are defined compositions of matter. The derivatives of the prior art are postulated to be aminoalkyl derivatives, specifically aminomethyl, but are not defined by any analytical means as to the site of substitution or the linker composition. The present invention is believed by the inventors to be the first disclosure of discreet hapten carboxyl derivatives of LSD substituted at the N-1 position.
Haptens are partial or incomplete antigens. They are protein-free substances, mostly low molecular weight substances, which are not capable of stimulating antibody formation, but which do react with antibodies. The latter are formed by coupling the hapten to a high molecular weight carrier and injecting this coupled product into humans or animals. Examples of haptens include therapeutic drugs such as digoxin and theophylline, drugs of abuse such as morphine and LSD, antibiotics such as gentamycin and vancomycin, hormones such as estrogen and progesterone, vitamins such as vitamin B12 and folic acid, thyroxin, histamine, serotonin, adrenaline and others.
A carrier, as the term is used herein, is an immunogenic substance, commonly a protein, that can join with a hapten, thereby enabling the hapten to stimulate an immune response. Carrier substances include proteins, glycoproteins, complex polysaccharides and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from the host.
An enzyme acceptor (EA) is an enzymatically inactive, polypeptide fragment of .beta.-galactosidase produced by a deletion mutant of the .beta.-galactosidase gene which, when combined or associated with an enzyme donor, is capable of forming active .beta.-galactosidase enzyme by the process of complementation.
An enzyme donor (ED) is an enzymatically inactive polypeptide fragment of .beta.-galactosidase comprising a peptide sequence capable of combining or associating with an enzyme acceptor to form active .beta.-galactosidase enzyme.
The term immunogenic as used herein refers to substances capable of producing or generating an immune response in an organism.
The term derivative refers to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions.
As used herein, a label or tracer is an identifying tag which, when attached to a carrier substance or molecule, can be used to detect an analyte. A label may be attached to its carrier substance directly or indirectly by means of a linking of bridging moiety. Examples of labels include enzymes such as .beta.-galactosidase and peroxidase, fluorescent compounds such as rhodamine and fluorescein isothiocyanate (FITC), luminescent compounds such as dioxetanes and luciferin, and radioactive isotopes such as .sup.125 I.
A peptide is any compound formed by the linkage of two or more amino acids by amide (peptide) bonds, usually a polymer of .alpha.-amino acids in which the a-amino group of each amino acid residue (except the NH.sub.2 -teraal) is linked to the .alpha.-arboxyl group of the next residue in a linear chain. The terms peptide, polypeptide and poly(amino acid) are use synonymously herein to refer to this class of compounds without restriction as to size. The largest members of this class are referred to as proteins.
A complex is a reversible association of chemical compounds or moieties held together by weak bonds or other forces, such as an enzyme-substrate complex (an association of an enzyme and one or more substrates that is the reacting moiety in an enzyme-catalyzed reaction), an antigen-antibody complex, a hapten-antibody complex, or an active enzyme complex of .beta.-galactosidase formed by complementation of an enzyme donor and an enzyme acceptor.